Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Vakonakis, Ioannis; Staunton, David; Ellis, Ian R.; Sarkies, Peter; Flanagan, Aleksandra; Schor, Ana M.; Schor, Seth L.; Campbell, Iain D.

doi:10.1074/jbc.m109.003673

Cited by 47 publications

(69 citation statements)

References 54 publications

(75 reference statements)

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“…Another example was provided by recent reports that III 3 spontaneously unfolds and binds anastellin, a mechanism that seems important for initiating or forming superFN aggregation (24,37,49). In the present study, we discovered that the instability of III 2 was also important for superFN-like aggregation of the small fragment III 1-2.…”

Section: Discussionsupporting

confidence: 71%

Fibronectin Aggregation and Assembly

Ohashi

Erickson

2011

Journal of Biological Chemistry

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The mechanism of fibronectin (FN) assembly and the selfassociation sites are still unclear and contradictory, although the N-terminal 70-kDa region ( I 1-9) is commonly accepted as one of the assembly sites. We previously found that I 1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that I 1-9 bound to the aggregate formed by anastellin and a small FN Fibronectin (FN)2 is a secreted protein expressed in the liver and circulated in blood as a soluble dimer (1). In addition to this plasma FN, some cell types, such as fibroblasts and endothelial cells, secrete FN and assemble it into extracellular matrix fibrils. FN is a modular protein containing 12 FN type I (FNI) domains, two FN type II domains, and 15-17 FN type III (FNIII) domains (2, 3). Two cysteines at the C terminus asymmetrically form interchain disulfide bonds (4, 5). Dimeric FN molecules interact with each other on the cell surface to form matrix fibrils. This process requires cell surface integrins (6 -9). Live cell imaging showed that integrin translocation by the actin cytoskeleton is crucial for the early stages of FN matrix assembly (10, 11). More recently, it has been reported that cell-to-cell adhesion via cadherin controls tissue tension and affects FN matrix formation during embryogenesis (12).The FN matrix had long been thought to be formed by disulfide-bonded FN multimers because the protein migrated at the top of an SDS gel under nonreducing conditions (13-16). However, studies by Chen and Mosher (17) A recent NMR study showed a unique structure for the domain pair III 1-2 (32). It had been recognized from sequence analysis that there is an 18-amino acid linker between these domains. The NMR structure showed that the A strand of III 2 was disordered, giving a total length of ϳ35 amino acids for the linker, and further indicated that III 1 and III 2 formed a closed compact structure, with a potential salt bridge between Lys-669 in III 1 and Asp-767 in III 2. These two amino acids were mutated to alanine, giving the mutant designated KADA. The native III 1-2 was found not to bind I 1-5, but the KADA mutation was reported to dramatically enhance binding of I 1-5, as measured by surface plasmon resonance.

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Section: Discussionsupporting

confidence: 71%

Fibronectin Aggregation and Assembly

Ohashi

Erickson

2011

Journal of Biological Chemistry

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“…Long range intramolecular interactions between 2-5 F1 and Fn type 3 modules (60) have recently been shown to mask motogenic sites within the collagen-binding domain of Fn. Changes in chemical shifts in the N-terminal strand (A strand) and E strands of F1 modules on F3 module binding (60) suggests that the F3-and SfbI-5-binding sites on 2-5 F1 partially overlap. The faster association rate observed for binding of wild-type SfbI-5 to pNTD compared with Fn (Table 3) is consistent with the FnBR-binding site being somewhat cryptic in Fn.…”

Section: Energy Of the Ntd/sfbi-5 Interaction Is Distributed Across Thementioning

confidence: 99%

Structural and Functional Analysis of the Tandem β-Zipper Interaction of a Streptococcal Protein with Human Fibronectin

Norris

Bingham

Harris

et al. 2011

Journal of Biological Chemistry

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Bacterial fibronectin-binding proteins (FnBPs) contain a large intrinsically disordered region (IDR) that mediates adhesion of bacteria to host tissues, and invasion of host cells, through binding to fibronectin (Fn). These FnBP IDRs consist of Fn-binding repeats (FnBRs) that form a highly extended tandem ␤-zipper interaction on binding to the N-terminal domain of Fn. Several FnBR residues are highly conserved across bacterial species, and here we investigate their contribution to the interaction. Mutation of these residues to alanine in SfbI-5 (a disordered FnBR from the human pathogen Streptococcus pyogenes) reduced binding, but for each residue the change in free energy of binding was <2 kcal/mol. The structure of an SfbI-5 peptide in complex with the second and third F1 modules from Fn confirms that the conserved FnBR residues play equivalent functional roles across bacterial species. Thus, in SfbI-5, the binding energy for the tandem ␤-zipper interaction with Fn is distributed across the interface rather than concentrated in a small number of "hot spot" residues that are frequently observed in the interactions of folded proteins. We propose that this might be a common feature of the interactions of IDRs and is likely to pose a challenge for the development of small molecule inhibitors of FnBP-mediated adhesion to and invasion of host cells.

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“…It is important to note that none of these bioactivities are manifested by any full-length fibronectin isoform, apparently as a consequence of steric hindrance. 31,32 Houard et al 29 have identified a second motif (HEEGH) in MSF module I-8 that seems to mediate the motogenic response of a breast cancer cell line (MCF7) and is additionally required for manifestation of its proteinase activity. Our recent data indicate that the motogenic response of certain target cells may be mediated by both IGD and HEEGH, whereas other cells only respond to the IGD motif.…”

Section: Msf: Molecular Characterisation and Diverse Functionalitymentioning

confidence: 99%